Long read sequencing technologies such as Oxford Nanopore (ONT) offer direct, simultaneous detection of DNA base modifications. The recent migration of ONT to the upgraded R10 chemistry has spurred the development of diverse methylation detection models. However, their performance and accuracy remain unclear. Here, leveraging diverse bacterial, plant, and mammalian datasets, we systematically evaluate the current landscape of tools and models for studying DNA methylation using nanopore sequencing. Our results demonstrate that the older models remain the best choice for studying CpG methylation. We note substantial improvement of newer tools in identifying 5-methylcytosine in non-CG contexts, 6-methyladenine, and 4-methylcytosine. We highlight the sensitivity of various tools to confounding methylation nearby. We also assess the computational performance of various tools, and effects of sequencing depth, methylation abundance, read quality, and basecalling mode. Our reusable pipelines and fully open access datasets provide a framework of resources to empower future benchmarking efforts. Our work thus details the strengths and limitations of the state-of-the-art methylation models and outlines practical guidelines for researchers using nanopore sequencing to study DNA modifications. ### Competing Interest Statement DTS has participated as a speaker in events organized by Oxford Nanopore Technologies. LZ is currently an employee of Oxford Nanopore Technologies. However, his association with this work predates his current employment. Other authors declare no competing interests. Rockefeller Foundation, https://ror.org/03sfkwk85, 2021 HTH 018 Department of Biotechnology, https://ror.org/03tjsyq23, BT/PR40264/BTIS/137/44/2022

YouTube video by BBC

Targeted DNA methylation editing is critical for establishing the causal relationship between DNA methylation and its function as well as for epigenetic crop breeding. Here, the authors develop a CRIS...

Research Laboratory Technician (Fixed Term) in the Department of Plant Sciences at the University of Cambridge.

Most crop plants, including sugar beet ( Beta vulgaris subsp. vulgaris ), suffer from domestication bottlenecks and low genetic diversity caused by extensive selection for few traits. However, crop wi...

Upon fertilization, male and female nuclei fuse to form the zygotic nucleus in angiosperms. Karyogamy is considered to be essential for proper embryogenesis; however, the transcriptional dynamics duri...

It is currently poorly understood how endophyte colonization and nematode parasitism affect host plants. To address this, we explored the transcriptio…

Published in Nineteenth-Century Contexts: An Interdisciplinary Journal (Ahead of Print, 2024)

Click on the article title to read more.